Abstract

BACKGROUND:

Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners.

RESULTS:

We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca(2+) influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca(2+)/CaM-dependent protein kinase IIα (CaMKII) a in Ca(2+)/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells.

CONCLUSIONS:

Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.

Rem2 overexpressed in neurons redistributes in response to neuronal stimulation.

(A) Images of neurons expressing either YFP or YFP-Rem2, before and after photoconductive stimulation, a non-invasive method of stimulating individual cells in a culture using light to target neurons grown on a silicon chip. To stimulate specific cells, we targeted the cell bodies of transfected neurons using a YFP filter, then stimulated the chip using 3–5 V of current for 2 msec duration at 20 Hz for 5 sec. Cells were imaged before and after stimulation to compare Rem2 distribution. Right panel is a magnification of the left panel to show details of Rem2 puncta. Scale bars represent 10 µm. (B) Normalized pixel value variance before stimulation, 1.0±0.26; after stimulation, 2.92±0.75, N = 15; paired t-test p = 0.006. Error bars represent SEM, and numbers within the bars indicate the number of cells averaged. (C) Images of neurons expressing either CFP or CFP-Rem2, before and after chemical stimulation. Individual cells in a culture of dissociated hippocampal neurons were imaged and their positions recorded, then the culture was stimulated with bath application of 25–100 µM glutamate/2.5–10 µM glycine. Follow-up images were taken of the recorded cells within 7 minutes of stimulation. Scale bars represent 10 µm. (D) Images such as in panel C were filtered and quantified by the variance in pixel values in each image. Variance was normalized to unstimulated cells. Ratio of CFP alone after/before stimulation, 1.10±0.07, N = 40; ratio of CFP-Rem2, 3.52±0.36, N = 39; t-test p<0.001. Numbers within the bars denote numbers of cells averaged for each bar. Error bars are SEM.

Top: Representation of the C-terminus of Rem2, with the putative CaM-binding determinant leucine residue highlighted in pink. (A) In the absence of stimulation, Rem2 residues 320–330 (regulatory region) may allosterically inhibit Rem2 association with a putative cytoskeletal regulatory element, and consequently no Rem2 puncta are observed. (B) Upon stimulation, Ca2+-CaM may bind the region around residue L317, allowing redistribution to occur by association with the cytoskeleton. (C) The Rem2 mutant 1–320 lacks the regulatory region. This would result in constitutive association with CaM and constitutive puncta. (D) The Rem2 mutant 1–310 lacks the CaM-binding region, and would not redistribute to puncta unless CaMKII is overexpressed. (E) Under basal conditions, Rem2 (blue circles) and CaMKII (red circles) are diffusely distributed in neurons. (F) Upon neuronal stimulation, Ca2+ influx through the NMDAR (black brackets) leads to aggregation of Rem2 and CaMKII, possibly at cytoskeletal elements (green lines).